The goal of this proposal is to understand the function of human homeodomain proteins and how they fit into the regulatory network of developmental gene expression. The importance of Drosophila homeobox-containing genes in controlling gene expression and embryonic development has been well established, and there is now extensive circumstantial evidence that mammalian genes containing the conserved 60 amino acid homeodomain play a similar role in mammals. Although numerous studies of the expression of homeodomain genes in mammalian embryos have correlated the expression of these genes with developmental regulation, their functions are still unknown. The objective of this proposal is to more directly assess the function of mammalian homeodomain proteins. We have demonstrated that the Hox 2. 1 (Hu 1) human homeodomain protein is a sequence-specific DNA binding protein, and that this protein acts as a site-dependent transcriptional activator in cotransfection assays. The specific aims of this proposal are: 1) Characterize the DNA binding specificity of the Hul protein, and the observed cooperative binding to adjacent sites. 2) Analyze the function of Hul as a transcription factor, including the promoter context and cell types that modulate its activity. The portions of the Hul protein responsible for its activities will be determined. 3). Identify target genes of the Hul protein by several techniques, including co-immunoprecipitation of genes with strong Hul binding sites, and by differential cDNA screening of genes whose expression is induced by the Hul protein. Much of the embryonic Hul gene expression is in the central nervous system, and the effect of Hul expression on potential target genes involved in neural differentiation will be examined. No target genes are known for mammalian homeodomain proteins, and the proposed identification of these target genes is an essential step in determining the role of the highly conserved homeodomain proteins in mammals. 4) As a model system for the complex embryonic regulation of transcription, characterize the regulatory elements that allow homeodomain gene expression in NT2/D1 teratocarcinoma cells only after retinoic acid induced differentiation. Analysis of homeodomain genes will serve to establish a starting point for the identification and characterization of the many other non-homeodomain genes that participate in the complex pathway of mammalian developmental regulation. The proposed analysis of the activities and targets of mammalian homeodomain proteins, in combination with the data available on embryonic expression patterns, will lead to a better understanding of the fundamental mechanisms that control human embryonic development.